Toxoplasmosis is one of the most common zoonoses in various regions of the world, being caused by the T. gondii protozoa. It is estimated that nearly one third of the world's population has been exposed to this pathogen. In Brazil, the prevalence of infection by T. gondii varies from 50 to 80%, with the highest rates occurring in some of the Northern and Southern states, and the lowest values in the Southeast states [Orefice, F. & Bonfioli, A. A. (2000). Uveìte Clìnica e Cir ùrgica(Uveitis and Surgical Clinic). In toxoplasmosis), pp. 619-680. Edited by E. C. Médica, Rio de Janeiro].
The T. gondii has three infectious forms: the tachyzoites, which multiply rapidly inside the cells throughout the body, the bradyzoites, which have a slower multiplication rate and are found in intracellular cysts in various tissues, and the sporozoites, which are present in the oocysts released in feces of the definitive hosts [Rey, L. (1991). Toxoplasma gondii e toxoplasmosis. In Parasitologia Humana. (Toxoplasma gondii and toxoplasmosis. In Human Parasitology.), pp. 274-285. Rio de Janeiro: Guanabara Koogan.]
An important characteristic of the T. gondii infection is the tissue cysts formation, during the chronic phase of disease. Cysts are rounded intracellular structures, bounded by an elastic wall formed by material derived from the host cell and the parasite. The cysts develop with higher frequency in the brain, eyes, cardiac and skeletal striated muscle, although its distribution and quantity vary between different species of hosts [Dubey, J. (1997). Tissue cyst tropism in Toxoplasma gondii: a comparison of tissue cyst formation in organs of cats, and rodents fed oocysts. Parasitology 115.]
T. gondii has an asexual reproduction stage and a sexual reproduction stage. The latter occurs only in Felidae, which leads to description of these mammals as the parasite's definitive hosts. [Tenter, A. M., Heckreroth, A. R. & Weiss, L. M. (2000). Toxoplasma gondii: from animals to humans. International Journal of Parasitology 30, 1217-1258.] The infection of intermediate and definitive hosts can occur through ingestion of tissue cysts in meat or viscera, by ingestion of oocysts sporulated from environmental contamination by feces of definitive hosts or also by transplacental transmission of tachyzoites [Tenter et. al., 2000].
Most of the toxoplasmosis cases in humans are asymptomatic. However, the acute infections in prenatal stage or during pregnancy can result in major complications. At pregnancy beginning, toxoplasmosis can cause abortion, neonatal death and fetal abnormalities. In the last three decades, the incidence of prenatal infection has been estimated in the range of 1 to 100 per 10,000 births, in different countries.
Regarding chronic infections in immunocompromised individuals, a reactivation can occur, triggering a pathological picture of great seriousness. For example, we can mention encephalitis, pulmonary toxoplasmosis, and disseminated toxoplasmosis, which have been observed in patients with immunodeficiencies related to various diseases such as Hodgkin's disease and AIDS. It is estimated that 40% of AIDS patients develop encephalitis caused by Toxoplasma, being that 10 to 30% die as result of this infection [Ferreira, M. S. & Borges, A. S. (2002). Some aspects of protozoan infections in immunocompromised patients—a review. Memórias do Instituto Oswaldo Cruz 97, 443-457] [Tenter et al., 2000]
The disseminated toxoplasmosis can also be a complication arising from organs and bone marrow transplant. This may arise from both the transplant of organs from donors infected with T. gondii to a susceptible recipient, as from reactivation of latent infection in recipients, due to immunosuppressive therapy [Tenter et al, 2000].
Considerable effort has been applied for developing a vaccine that prevents the transmission of T. gondii within human and animal populations. The main objective of a vaccine against T. gondii is to generate an immune response in different hosts, controlling the parasite's replication and its transmission within the population. The induction of an immune response, prior to infection with the parasite, would prevent the release of oocysts by definitive hosts, preventing the formation of tissue cysts in intermediate hosts, and their oral transmission via meat, and would prevent the development of a state of asymptomatic carriers subject to a recall in humans. A vaccine could also prevent the acute toxoplasmosis in pregnant women and the transplacental transmission [A lexander, J., Jebbari, H., Bluethmann, H., Satoskar, A. & Roberts, C. W. (1996). Immunological control of Toxoplasma gondii and appropriate vaccine design. Current Topics in Microbiology and Immunology: Toxoplasma gondii., 183-195].
Initial studies showed that it is possible to induce protective immunity, with activation of T CD4+, T CD8+ cells and production of IFN-γ, using attenuated forms of the parasite. However, vaccines produced this way are not suitable for application in humans, considering the risk of reversion of the vaccine samples to a pathogenic state [Gazzinelli, R. T., Hakim, F. T., Hieny, S., Shearer, G. M. & Sher, A. (1991). Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. Journal of Immunology 146, 286-292] [Hiramoto, R. M., Galisteo, A. J., Nascimento, N. & Andrade Jr, H. F. (2002). 200Gy sterilized Toxoplasma gondii tachyzoites maintain metabolic functions and mammalian cell invasion, eliciting cellular immunity and cytokine response similar to natural infection in mice. Vaccine 20, 2072-2081] [Rodrigues, M. M., Boscardin, S. B., Vasconcelos, J. R., Hiyane, M. I., Salay, G. & Soares, I. S. (2003). Importance of CD8 T cell-mediated immune response during intracellular parasitic infections and its implications for the development of effective vaccines. Annals of the Brazilian Academy of Sciences 75, 443-468] [Sayles, P. & Johnson, L. L. (1996). Intact immune defenses are required for mice to resist the ts-4 vaccine strain of Toxoplasma gondii. Infection & Immunity 64, 3088-3092]. To bypass this problem, molecular biology techniques that allow the parasite genes cloning and expression has been used to develop the named subunit vaccine, containing only T. gondii immunogenic components. (Biemans et al., 1998, Büllow & Boothroyd, 1991). (Biemans et al., 1998, Bulow & Boothroyd, 1991).
Recently, significant progress has been made in identifying T. gondii antigens that can induce protective immune response. Most of the work has been focused on surface antigens expressed in tachyzoites, with particular interest in SAG1, SAG2 and SAGS, which act in the host cell invasion process [Lekutis, C., Fergunson, D. J. P., Grigg, M. E., Camps, M. & Boothroyd, J. C. (2001). Surface antigens of Toxoplasma gondii: variations on a theme. International Journal of Parasitology 67, 5869-5876]. The SAGs sequence are maintained within different strains of T. gondii and, moreover, these antigens stimulate cellular and humoral response, which makes these proteins good candidates for development of vaccines.
Amongst vectors for expression of these antigens, both in humans as in other animals, the recombinant viruses deserve mention [Rocha, C., Caetano, B., Machado, A. & Bruna-Romero, O. (2004). Recombinant viruses as tools to induce protective cellular immunity against infectious diseases. International Microbiology 7, 89-94] [Rodrigues et al., 2003]. Studies using different recombinant virus (vaccinia, adenovirus, Sindbis and influenza) expressing the Plasmodium yoelii antigens, showed that such viruses are capable of inducing a protective immunity in BALB/c mice exposed to an experimental infection by P. yoelii sporozoites [Li, S., Rodrigues, M., Rodriguez, D., Rodriguez, J. R., Esteban, M., Palese, P., Nussenzweig, R. S., Zavala, F. & Alexander, D. J. (1993b). Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria. Proceedings of the National Academy of Sciences of the United States of America 90, 5214-8] [Rodrigues, M., Li, S., Murata, K., Rodriguez, D., Rodriguez, J. R., Bacik, I., Bennink, J. R., Yewdell, J. W., Garcia-Sastre, A. & Nussenzweig, R. S. (1994). Influenza and vaccinia viruses expressing malaria CD8+ T and B cell epitopes. Comparison of their immunogenicity and capacity to induce protective immunity. Journal of Immunology 153, 4636-48] [Tsuji, M., Bergmann, C., Takita-Sonoda, Y., Murata, K., Rodrigues, E., Nussenzweig, R. & Zavala, F. (1998). Recombinant Sindbis viruses expressing a cytotoxic T-lymphocyte epitope of a malaria parasite or of influenza virus elicit protection against the corresponding pathogen in mice. Journal of Virology 72, 6907-6910].
Most of the experimental data obtained so far showed that, when the viral vectors are used alone, the protection level achieved was significantly lower than that observed when two different viral vectors expressing the same antigen were used for prime and boost immunization. [Gherardi, M. M., Najera, J. L., Perez-Jimenez, E., Guerra, S., Garcia-Sastre, A. & Esteban, M. (2003). Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes. Journal of Virology 77, 7048-7057] [Li, S., Polonis, V., Isobe, H., Zaghouani, H., Guinea, R., Moran, T., Bona, C. & Palese, P. (1993a). Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1. Journal of Virology 67, 6659-66] [Murata, K., Garcia-Sastre, A., Tsuji, M., Rodrigues, M., Rodriguez, D., Rodriguez, J. R., Nussenzweig, R. S., Palese, P., Esteban, M. & Zavala, F. (1996). Characterization of in vivo primary and secondary CD8+ T cell responses induced by recombinant influenza and vaccinia viruses. Cellular Immunology 173, 96-107] [Shiver, J. W., Fu, T. M., Chen, L., Casimiro, D. R., Davies, M. E., Evans, R. K., Zhang, Z. Q., Simon, A. J., Trigona, W. L., Dubey, S. A., Huang, L., Harris, V. A., Long, R. S., Liang, X., Handt, L., Schleif, W. A., Zhu, L., Freed, D. C., Persaud, N. V., Guan, L., Punt, K. S., Tang, A., Chen, M., Wilson, K. A., Collins, K. B., Heidecker, G. J., Fernandez, V. R., Perry, H. C., Joyce, J. G., Grimm, K. M., Cook, J. C., Keller, P. M., Kresock, D. S., Mach, H., Troutman, R. D., Isopi, L. A., Williams, D. M., Xu, Z., Bohannon, K. E., Volkin, D. B., Montefiori, D. C., Miura, A., Krivulka, G. R., Lifton, M. A., Kuroda, M. J., Schmitz, J. E., Letvin, N. L., Caulfield, M. J., Bett, A. J., Youil, R., Kaslow, D. C. & Emini, E. A. (2002). Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 415, 331-5].
This kind of immunization strategy is known as heterologous system for priming and boosting the immune response. As an example on the effectiveness of this strategy, we could mention the total inhibition of development of hepatic forms and 100% protection against malaria, in mice that were first immunized with recombinant adenovirus expressing the P. yoelii or P. berghei CS protein and boosted with a vaccinia virus expressing this same protein [Bruna-Romero, O., Gonzalez-Aseguinolaza, G., Hafalla, J. C., Tsuji, M. & Nussenzweig, R. S. (2001). Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen. Proceeding of National Academy of Sciences of United States of America 98, 11491-11496] [Gilbert, S. C., Schneider, J., Hannan, C. M., Hu, J. T., Plebanski, M., Sinden, R. & Hill, A. V. (2002). Enhanced CD8 T cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunization regimes. Vaccine 20, 1039-1045].
Amongst viruses potentially usable as antigens delivery vectors, the adenoviruses, the modified vaccinia Ankara viruses (MVA) and the influenza viruses have some characteristics making them interesting candidates for use in induction of a heterospecific immune response against T. gondii and other pathogens [Garcia-Sastre, A. (2000). Transfectant influenza viruses as antigen delivery vectors. Advances in Virus Research 55, 579-97] [Rocha et al., 2004] [Tatsis, N. & Ertl, H. C. (2004). Adenoviruses as vaccine vectors. Molecular Therapy 10, 616-629].
Amongst advantages of using the influenza virus, we could mention the fact that it does not integrate in the host genome and does not persist in the body. Furthermore, the existence of different influenza virus variants and subtypes enables the execution of sequential immunizations using two subtypes or variants of this virus [Ferko, B., Stasakova, J., Sereinig, S., Romanova, J., Katinger, D., Niebler, B., Katinger, H. & Egorov, A. (2001). Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice. Journal of Virology 75, 8899-908]. Another favorable argument for choosing the influenza virus as vector for expression of heterologous sequences consists in that currently the molecular biology techniques for genetic manipulation of this virus are already well developed [Neumann, G. & Kawaoka, Y. (2002). Generation of influenza A virus from cloned cDNAs—historical perspective and outlook for the new millennium. Reviews in Medical Virology 12, 13-30] [Neumann, G. & Kawaoka, Y. (2004). Reverse genetics systems for the generation of segmented negative-sense RNA viruses entirely from cloned cDNA. Current Topics in Microbiology and Immunology 283, 43-60] and different strategies already exist for expression of heterologous proteins using recombinant influenza viruses. [Garcia-Sastre et al., 1994] [Machado, A., Naffakh, N., van der Werf, S. & Escriou, N. (2003). Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter. Virology 313, 235-249] [Percy, N., Barclay, W. S., Garcia Sastre, A. & Palese, P. (1994). Expression of a foreign protein by influenza A virus. Journal of Virology 68, 4486-4492] [Takasuda, N., Enami, S., Itamura, T. & Takemori (2002). Intranasal inoculation of a recombinant influenza virus containing exogenous nucleotides in the NS segment induces mucosal immune response against the exogenous gene product in mice. Vaccine 20, 1579-1585] [Watanabe, T., Watanabe, S., Noda, T., Fujii, Y. & Kawaoka, Y. (2003). Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes. Journal of Virology 77, 10575-83].
Additional to the advantages above mentioned, is the ability of this of vector type to induce an heterospecific immune response, both local as systemic [Garulli, B., Kawaoka, Y. & Castrucci, M., R. (2004). Mucosal and systemic immune responses to a human immunodeficiency virus type 1 epitope induced upon vaginal infection with a recombinant influenza A virus. Journal of Virology 78, 1020-1025] [Gherardi, M. M., Najera, J. L., Perez-Jimenez, E., Guerra, S., Garcia-Sastre, A. & Esteban, M. (2003). Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes. Journal of Virology 77, 7048-7057]. The existence of a well-developed technology for influenza virus industrial scale production and the existence of attenuated viral strains are additional favorable points in using this vector. Regarding the attenuated strains, their use in humans has been authorized in the United States and they could then be used to construct recombinant live vaccines, able to immunize simultaneously against the influenza virus and some other pathogen [Izurieta, H. S., Haber, P., Wise, R. P., Iskander, J., Pratt, D., Mink, C., Chang, S., Braun, M. M. & Ball, R. (2005). Adverse events reported following live, cold-adapted, intranasal influenza vaccine. Jama 294, 2720-5] [Subbarao, K. & Katz, J. M. (2004). Influenza vaccines generated by reverse genetics. Curr Top Microbiol Immunol 283, 313-42].
Adenoviruses, in turn, are genetically stable, easy to handle and possess a remarkable capacity for heterologous sequences expression. Adenoviruses are still characterized by multiplying well in cellular culture and having broad tropism, being able to infect a wide range of cell types (replicating and non-replicating ones) and tissues. Moreover, they can be inoculated both by systemic and mucous routes.
Another favorable aspect of adenoviral vectors consists in that they are relatively safe, since they do not integrate in the host cells chromosomes [Tatsis & Ertl, 2004], and in that diseases caused by serotypes that normally infect humans are of little significance. Moreover, it is possible deleting significant portions of the viral genome, resulting in defective vectors for replication. This contributes not only to further enhance the security of this type of vector, but also to allow a better heterologous antigen expression [Xiang, Z. & Ertl, H. C. (1999). Induction of mucosal immunity with a replication-defective adenoviral recombinant. Vaccine 17, 2003-2008] [Xiang, Z. Q., Yang, Y., Wilson, J. M. & Ertl, H. C. (1996). A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier. Virology 219, 220-227].
Another interesting aspect of adenoviruses is the ability to change their cellular tropism by modifying the protein responsible for the virus adherence to host cells [Tatsis & Ertl, 2004]. Finally, it is possible using adenoviral vectors based on serotypes of low circulation in human population, or even non-human serotypes [Barouch, D. H., Pau, M. G., Custers, J. H., Koudstaal, W., Kostense, S., Havenga, M. J., Truitt, D. M., Sumida, S. M., Kishko, M. G., Arthur, J. C., Korioth-Schmitz, B., Newberg, M. H., Gorgone, D. A., Lifton, M. A., Panicali, D. L., Nabel, G. J., Letvin, N. L. & Goudsmit, J. (2004). Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity. Journal Immunology 172, 6290-6297] [Farina, S. F., Gao, G. P., Xiang, Z. Q., Rux, J. J., Burnett, R. M., Alvira, M. R., Marsh, J., Ertl, H. C. & Wilson, J. M. (2001). Replication-defective vector based on a chimpanzee adenovirus. Journal of Virology 75, 11603-11613] [Fitzgerald, J., Gao, G., Reyes-Sandoval, A., Pavlakis, G., Xiang, Z., Wlazlo, A., Giles-Davis, W., Wilson, J. & Ertl, H. (2003). A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag. Journal Immunology 170, 1416-1422] [Reyes-Sandoval, A., Fitzgerald, J. C., Grant, R., Roy, S., Xiang, Z. Q., Li, Y., Gao, G. P., Wilson, J. M. & Ertl, H. C. (2004). Human immunodeficiency virus type 1-specific immune responses in primates upon sequential immunization with adenoviral vaccine carriers of human and simian serotypes. Journal of Virology 78, 7392-7399], thus, allowing to bypass potential problems related to occurrence of a pre-existing immune response against the adenoviral vector.
Historically, the MVA (from “modified vaccinia virus Ankara) was created to serve as a safe vaccine against smallpox. This virus was attenuated after more than 570 passages on primary chicken embryo fibroblasts (CEF) cultures, during which occurred the deletion of several of the vaccinia virus genes responsible for virulence, rendering them unable to generate productive infections in mammals cells [Sutter, G. & Staib, C. (2003). Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery. Curr Drug Targets Infect Disord 3, 263-71]. The MVA viruses have the following advantages concerning its use as vaccine vectors:                (1) Ability to receive the insertion of large exogenous DNA sequences (up to 25 kb).        (2) Stability to freezing and freeze-drying.        (3) Lack of integration in the host genome due to the fact that this virus multiplies in the infected cells cytoplasm.        (4) Ability to induce cellular and humoral immune response against the heterologous antigen [Rocha et al., 2004].        